Identification of Novel tet(X3) Variants Resistant To Tigecycline in Acinetobacter Species

ABSTRACT The emergence of the tet(X) gene is a severe challenge to global public health security, as clinical tigecycline resistance shows a rapidly rising trend. In this research, we identified two tigecycline-resistant Acinetobacter sp. strains containing seven novel tet(X3) variants recovered from fecal samples from Chinese farms. The seven Tet(X3) variants showed 15.4% to 99.7% amino acid identity with Tet(X3). By expressing tet(X3.7) and tet(X3.9), the tigecycline MIC values for Escherichia coli JM109 increased 64-fold (from 0.13 to 8 mg/L). However, the other tet(X3) variants did not have a significant change in the MIC of tigecycline. We found that the 26th amino acid site of Tet(X3.7) changed from proline to serine, and the 25th amino acid site of Tet(X3.9) changed from glycine to alanine, which reduced the MIC of tigecycline by 2-fold [the MIC of tet(X3) to tigecycline was 16 mg/L] but did not affect its expression to tigecycline. The tet(X3) variants surrounded by mobile genetic elements appeared in the structure of gene clusters with tandem repeat sequences and were adjacent to the site-specific recombinase-encoding gene xerD. Therefore, there is a risk of horizontal transfer of resistant genes. Our study reports seven novel tet(X3) variants; the continuing emergence of tigecycline variants makes continuous monitoring of resistance to tigecycline even more critical. IMPORTANCE Although it is illegal to use tigecycline and carbapenems to treat bacterial infections in animals, we can still isolate bacteria containing both mobile resistance genes from animals, and tet(X) is currently an essential factor in degrading tigecycline. Here, we characterized two multidrug-resistant Acinetobacter sp. strains that contained vital resistance genes, such as sul2, a blaOXA-164-like gene, floR, tetM, and multiple novel tet(X3) variants with different tandem structures. It is of paramount significance that their mechanism may transfer to other Gram-negative pathogens, even if their tandem structures have no cumulative effect on tigecycline resistance.

The manuscript identified seven tet(X3) variants, designated from tet(X3.3) to tet(X3.9), however their amino acid identities compared to tet(X3) were 58.78% 66.13% 22.71 % 23.28% 99.7% 15.4% and 99.7%, respectively. Can the amino acid sequences with 15.4%-58.78% homology be named as variants of tet(X3)? The authors need collect all tetX variants to construct a phylogenetic tree for more accurate and scientific naming? Moreover, the English grammar in the paper is rather bad and should be improved. I will suggest the authors consult a native speaker to help you remove the flaws from the manuscript. Line 127-135: It is confusing and should reconstruct the sentence. Line 151: The word "brone" should be corrected to "borne". Line 157-159: It is confusing and should reconstruct the sentence. Line 173: E.coil should be E.coli Line 174: The word "brone" looks like a spelling mistake ? Lines 204-205: "Nanopore and Illumina reads were combined to produce data for genome assembly were performed with Unicycler version 0.4.3" The complete sequence of the strain in the manuscript is the result of assembly by the unicycler software, however its sequence contains multiple tandem repeat structures, due to the specificity of this structure (the assembly is prone to mismatches), whether the authors used other means (such as checking the long reads raw data or PCR products) to verify the accuracy of these structures? 1.Currently, there are no MIC breakpoints for tigecycline against Acinetobacter spp in EUCAST, CLSI100, and FDA standards. EUCAST and FDA Antibacterial Susceptibility Test Interpretive Criteria contains tigecycline breakpoints for Enterobacteriaceae. Acinetobacter spp refer to this Criteria (tigecycline breakpoints for Enterobacteriaceae) temporarily. The reference for tigecycline's breakpoint judgment for Acinetobacter spp can be described more clearly.
2.According to the drug resistance rate and detection rate reported in the literature, Acinetobacter baumannii has received the highest clinical attention among Acinetobacter spp. It may be more clinically meaningful to study the novel tet(X3) variants and whether these genes can be conjugated to A. baumannii. Of course, It cannot be excluded that other species within the Acinetobacter genus would be the main prevalent species for future infections.
3.As your described, the novel tet(X3) variants could not conjugate transferred with E. coli EC600 and J53 after three repeats. In the natural state, whether novel tet(X3) variants could conjugated with Enterobacteriaceae or acinetobacter baumannii may require further study.

Reviewer 3:
The manuscript identified seven tet(X3) variants, designated from tet(X3.3) to tet(X3.9), however their amino acid identities compared to tet(X3) were 58.78% 66.13% 22.71 % 23.28% 99.7% 15.4% and 99.7%, respectively. Can the amino acid sequences with 15.4%-58.78% homology be named as variants of tet(X3)? The authors need collect all tetX variants to construct a phylogenetic tree for more accurate and scientific naming? Moreover, the English grammar in the paper is rather bad and should be improved. I will suggest the authors consult a native speaker to help you remove the flaws from the manuscript.

Other comments:
Line 35: clinical should be clinic.  Line 127-135: It is confusing and should reconstruct the sentence.
Line 157-159: It is confusing and should reconstruct the sentence.
Line 173: E. coil should be E. coli Line 174: The word "brone" looks like a spelling mistake? Lines 204-205: "Nanopore and Illumina reads were combined to produce data for genome assembly were performed with Unicycler version 0.4.3" The complete sequence of the strain in the manuscript is the result of assembly by the unicycler software, however its sequence contains multiple tandem repeat structures, due to the specificity of this structure (the assembly is prone to mismatches), whether the authors used other means (such as checking the long reads raw data or PCR products) to verify the accuracy of these structures?   Acinetobacter spp refer to this Criteria (tigecycline breakpoints for Enterobacteriaceae) temporarily. The reference for tigecycline's breakpoint judgment for Acinetobacter spp can be described more clearly.

Response to Reviewers
As suggested by the reviewer, the content of "Antimicrobial susceptibility testing" has been modified, and the modifications can be seen on lines 199-202.
2.According to the drug resistance rate and detection rate reported in the literature, Acinetobacter baumannii has received the highest clinical attention among Acinetobacter spp. It may be more clinically meaningful to study the novel tet(X3) variants and whether these genes can be conjugated to A. baumannii. Of course, It cannot be excluded that other species within the Acinetobacter genus would be the main prevalent species for future infections.
According to the drug resistance rate and detection rate reported in the literature, Acinetobacter baumannii does receive the highest clinical attention. Since our research group did not have a suitable A. baumannii as the recipient strain, we were unable to investigate whether the tet(X3) variants could be conjugated to A. baumannii. Meanwhile, according to the method of "genetic diversity and characteristics of high-level tigecycline resistance Tet(X) in Acinetobacter species", the novel tet(X3) variants could be successfully transferred into A. baylyi ADP1, which also has some clinically meaningful.
3.As your described, the novel tet(X3) variants could not conjugate transferred with E. coli EC600 and J53 after three repeats. In the natural state, whether novel tet(X3) variants could conjugated with Enterobacteriaceae or acinetobacter baumannii may require further study.
In this research, the novel tet(X3) variants could not conjugate transferred with E. coli EC600 and J53 after three repeats. In the natural state, whether novel tet(X3) variants could conjugated with Enterobacteriaceae or Acinetobacter baumannii may require further study. Moreover, the English grammar in the paper is rather bad and should be improved. I will suggest the authors consult a native speaker to help you remove the flaws from the manuscript.
As suggested by the reviewer, the use of English language throughout the article has been revised.
The "Acinetobacter Bailey ADP1" that appears throughout the article has also been changed to "Acinetobacter Baylyi ADP1".

Other comments:
Line 35: clinical should be clinic.
As suggested by the reviewer, the content of "Introduction" has been modified, the "clinical" has been changed to "clinic", and the modifications can be seen on lines 35.

Line 47: The reference for tet(M) variant was not cited here.
As suggested by the reviewer, the content of "Introduction" has been modified, and the modifications can be seen on lines 46-48 and Ref. 26.

Line 48:enzyme should be enzemy
As suggested by the reviewer, the "enzyme" that appears throughout the article has been changed to "enzymes", and the modifications can be seen on lines 48. The authors should describe this in the text in detail.
As suggested by the reviewer, we sequenced the transconjugants JAT2044 and JAT2091 and found that 5,117-bp circular intermediate (res-ISCR2-ISVsa3-xerD-tet(X3)) forming a minicircle plasmid in the transconjugants JAT2044 to mediates the resistance of the tigecycline. And part of the region of pBDT2091-4 plasmid was re-formed a 27,995-bp plasmid and named pJAT2091.
Line 79: The authors should check 13 consecutive tet(X3) copies carefully to confirm whether it is correct using the original sequencing data.
As suggested by the reviewer, we performed confirmation, but due to the large repeat sequences, we could only confirm that up to 7 consecutive tet(X3) copies results were correct using the raw sequencing data, and finally confirmed that 13 consecutive tet(X3) copies structures were correct by sequence splicing the results. As suggested by the reviewer, this sentence has been revised, and the modifications can be seen on lines 140-144.
As suggested by the reviewer, the "brone" that appears throughout the article has been changed to "borne", and the modifications can be seen on lines 158-160.
Line 157-159: It is confusing and should reconstruct the sentence.
As suggested by the reviewer, this sentence has been revised, and the modifications can be seen on lines 163-168.

Line 173: E.coil should be E.coli
As suggested by the reviewer, the "E.coil" that appears throughout the article has been changed to "E.coli", and the modifications can be seen on lines 180-182.

Line 174: The word "brone" looks like a spelling mistake ?
As suggested by the reviewer, the "brone" that appears throughout the article has been changed to "borne", and the modifications can be seen on lines 158-160.
Lines 204-205: "Nanopore and Illumina reads were combined to produce data for genome assembly were performed with Unicycler version 0.4.3" The complete sequence of the strain in the manuscript is the result of assembly by the unicycler software, however its sequence contains multiple tandem repeat structures, due to the specificity of this structure (the assembly is prone to mismatches), whether the authors used other means (such as checking the long reads raw data or PCR products) to verify the accuracy of these structures?
As suggested by the reviewer, the results of genome assembly with Unicycler version 0.4.3 were quality-checked. We also verify the accuracy of these structures by examining the long read raw data, but due to the high similarity between the tet(X3) variant sequences, it was inconvenient to verify them by PCR amplification. As suggested by the reviewer, the content of "references" has been modified, and the format of references has been uniformly modified to use the ASM literature format.
In addition to this, english language, each corresponding figure and reference has been modified according to the revision of the content in the manuscript. Thank you for submitting your manuscript to Microbiology Spectrum. When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues raised by the reviewers as file type "Response to Reviewers," not in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only". Please use this link to submit your revised manuscript -we strongly recommend that you submit your paper within the next 60 days or reach out to me. Detailed instructions on submitting your revised paper are below.

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To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER. • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file. • Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
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Response to Reviewers (Modifications in the manuscript are highlighted in yellow)
Questions: Reviewer 1: 1.Currently, there are no MIC breakpoints for tigecycline against Acinetobacter spp in EUCAST, CLSI100, and FDA standards. EUCAST and FDA Antibacterial Susceptibility Test Interpretive Criteria contains tigecycline breakpoints for Enterobacteriaceae. Acinetobacter spp refer to this Criteria (tigecycline breakpoints for Enterobacteriaceae) temporarily. The reference for tigecycline's breakpoint judgment for Acinetobacter spp can be described more clearly.
2.According to the drug resistance rate and detection rate reported in the literature, Acinetobacter baumannii has received the highest clinical attention among Acinetobacter spp. It may be more clinically meaningful to study the novel tet(X3) variants and whether these genes can be conjugated to A. baumannii. Of course, It cannot be excluded that other species within the Acinetobacter genus would be the main prevalent species for future infections.
3.As your described, the novel tet(X3) variants could not conjugate transferred with E. coli EC600 and J53 after three repeats. In the natural state, whether novel tet(X3) variants could conjugated with Enterobacteriaceae or acinetobacter baumannii may require further study.

Reviewer 3:
The manuscript identified seven tet(X3) variants, designated from tet(X3.3) to tet(X3.9), however their amino acid identities compared to tet(X3) were 58.78% 66.13% 22.71 % 23.28% 99.7% 15.4% and 99.7%, respectively. Can the amino acid sequences with 15.4%-58.78% homology be named as variants of tet(X3)? The authors need collect all tetX variants to construct a phylogenetic tree for more accurate and scientific naming? Moreover, the English grammar in the paper is rather bad and should be improved. I will suggest the authors consult a native speaker to help you remove the flaws from the manuscript.

Other comments:
Line 35: clinical should be clinic.  As suggested by the reviewer, the content of "Antimicrobial susceptibility testing" has been modified, and the modifications can be seen on lines 201-204.

Acinetobacter baumannii has received the highest clinical attention among Acinetobacter spp.
It may be more clinically meaningful to study the novel tet(X3) variants and whether these genes can be conjugated to A. baumannii. Of course, It cannot be excluded that other species within the Acinetobacter genus would be the main prevalent species for future infections.
According to the drug resistance rate and detection rate reported in the literature, Acinetobacter baumannii does receive the highest clinical attention. As suggested by the reviewer to conduct the study, the strains could not be successfully transferred into the recipient Acinetobacter baumannii, the modifications can be seen on lines 116-118, 208-211, 245-248. Meanwhile, according to the methodology of the literature "genetic diversity and characteristics of high-level tigecycline resistance Tet(X) in Acinetobacter species", the novel tet(X3) variants could be successfully transferred into A. baylyi ADP1, which also has some clinically meaningful.

Moreover, the English grammar in the paper is rather bad and should be improved. I will suggest the authors consult a native speaker to help you remove the flaws from the manuscript.
As suggested by the reviewer, the use of English language throughout the article has been revised.
The "Acinetobacter Bailey ADP1" that appears throughout the article has also been changed to "Acinetobacter Baylyi ADP1".

Other comments:
Line 35: clinical should be clinic.
As suggested by the reviewer, the content of "Introduction" has been modified, the "clinical" has been changed to "clinic", and the modifications can be seen on lines 36.

Line 47: The reference for tet(M) variant was not cited here.
As suggested by the reviewer, the content of "Introduction" has been modified, and the modifications can be seen on lines 47-48 and Ref. 26.

Line 48:enzyme should be enzemy
As suggested by the reviewer, the "enzyme" that appears throughout the article has been changed to "enzymes", and the modifications can be seen on lines 49. Line 79: The authors should check 13 consecutive tet(X3) copies carefully to confirm whether it is correct using the original sequencing data.
As suggested by the reviewer, we performed confirmation, but due to the large repeat sequences, we could only confirm that up to 7 consecutive tet(X3) copies results were correct using the raw sequencing data, and finally confirmed that 13 consecutive tet(X3) copies structures were correct by sequence splicing the results. As suggested by the reviewer, the content of "the blaOXA-276 related part including Fig 5" have been deleted.

Line 127-135: It is confusing and should reconstruct the sentence.
As suggested by the reviewer, this sentence has been revised, and the modifications can be seen on lines 142-146.
As suggested by the reviewer, the "brone" that appears throughout the article has been changed to "borne", and the modifications can be seen on lines 161.
Line 157-159: It is confusing and should reconstruct the sentence.
As suggested by the reviewer, this sentence has been revised, and the modifications can be seen on lines 165-170.

Line 173: E.coil should be E.coli
As suggested by the reviewer, the "E.coil" that appears throughout the article has been changed to "E.coli", and the modifications can be seen on lines 183.

Line 174: The word "brone" looks like a spelling mistake ?
As suggested by the reviewer, the "brone" that appears throughout the article has been changed to "borne", and the modifications can be seen on lines 161. Lines 204-205: "Nanopore and Illumina reads were combined to produce data for genome assembly were performed with Unicycler version 0.4.3" The complete sequence of the strain in the manuscript is the result of assembly by the unicycler software, however its sequence contains multiple tandem repeat structures, due to the specificity of this structure (the assembly is prone to mismatches), whether the authors used other means (such as checking the long reads raw data or PCR products) to verify the accuracy of these structures?
As suggested by the reviewer, the results of genome assembly with Unicycler version 0.4.3 were quality-checked. We also verify the accuracy of these structures by examining the long read raw data, but due to the high similarity between the tet(X3) variant sequences, it was inconvenient to verify them by PCR amplification. As suggested by the reviewer, the content of "references" has been modified, and the format of references has been uniformly modified to use the ASM literature format.
In addition to this, english language, each corresponding figure and reference has been modified according to the revision of the content in the manuscript. Quality of English grammar and style is still not sufficient! Please revise accordingly, I would highly recommend a professional editing service. Also, please explain why most of your conjugation experiments did not work. You might even want to consider including this in the discussion section.
Thank you for submitting your manuscript to Microbiology Spectrum. As you will see your paper is very close to acceptance. Please modify the manuscript along the lines I have recommended. As these revisions are quite minor, I expect that you should be able to turn in the revised paper in less than 30 days, if not sooner. If your manuscript was reviewed, you will find the reviewers' comments below.
When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues raised by the reviewers as file type "Response to Reviewers," not in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only". Please use this link to submit your revised manuscript. Detailed instructions on submitting your revised paper are below.

Link Not Available
Thank you for the privilege of reviewing your work. Below you will find instructions from the Microbiology Spectrum editorial office and comments generated during the review.
The ASM Journals program strives for constant improvement in our submission and publication process. Please tell us how we can improve your experience by taking this quick Author Survey. Sincerely,

Katharina Schaufler
Editor, Microbiology Spectrum Reviewer comments:

Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website.
Corresponding authors may join or renew ASM membership to obtain discounts on publication fees. Need to upgrade your membership level? Please contact Customer Service at Service@asmusa.org.
Thank you for submitting your paper to Microbiology Spectrum.

Response to Reviewers (Modifications in the manuscript are highlighted in yellow)
Questions: Reviewer 1: 1.Currently, there are no MIC breakpoints for tigecycline against Acinetobacter spp in EUCAST, CLSI100, and FDA standards. EUCAST and FDA Antibacterial Susceptibility Test Interpretive Criteria contains tigecycline breakpoints for Enterobacteriaceae. Acinetobacter spp refer to this Criteria (tigecycline breakpoints for Enterobacteriaceae) temporarily. The reference for tigecycline's breakpoint judgment for Acinetobacter spp can be described more clearly.
2.According to the drug resistance rate and detection rate reported in the literature, Acinetobacter baumannii has received the highest clinical attention among Acinetobacter spp. It may be more clinically meaningful to study the novel tet(X3) variants and whether these genes can be conjugated to A. baumannii. Of course, It cannot be excluded that other species within the Acinetobacter genus would be the main prevalent species for future infections.
3.As your described, the novel tet(X3) variants could not conjugate transferred with E. coli EC600 and J53 after three repeats. In the natural state, whether novel tet(X3) variants could conjugated with Enterobacteriaceae or acinetobacter baumannii may require further study.

Reviewer 3:
The manuscript identified seven tet(X3) variants, designated from tet(X3.3) to tet(X3.9), however their amino acid identities compared to tet(X3) were 58.78% 66.13% 22.71 % 23.28% 99.7% 15.4% and 99.7%, respectively. Can the amino acid sequences with 15.4%-58.78% homology be named as variants of tet(X3)? The authors need collect all tetX variants to construct a phylogenetic tree for more accurate and scientific naming? Moreover, the English grammar in the paper is rather bad and should be improved. I will suggest the authors consult a native speaker to help you remove the flaws from the manuscript.

Other comments:
Line 35: clinical should be clinic.

Editor reviews:
Please explain why most of your conjugation experiments did not work. You might even want to consider including this in the discussion section. As suggested by the reviewer, the content of "Antimicrobial susceptibility testing" has been modified, and the modifications can be seen on lines 212-215.

Response to Reviewers
2.According to the drug resistance rate and detection rate reported in the literature, Acinetobacter baumannii has received the highest clinical attention among Acinetobacter spp.
It may be more clinically meaningful to study the novel tet(X3) variants and whether these genes can be conjugated to A. baumannii. Of course, It cannot be excluded that other species within the Acinetobacter genus would be the main prevalent species for future infections.
According to the drug resistance rate and detection rate reported in the literature, Acinetobacter baumannii does receive the highest clinical attention. As suggested by the reviewer to conduct the study, the strains could not be successfully transferred into the recipient Acinetobacter baumannii, the modifications can be seen on lines 118-120, 219-222, 262-264. Meanwhile, according to the methodology of the literature "Genetic diversity and characteristics of high-level tigecycline resistance Tet(X) in Acinetobacter species", the novel tet(X3) variants could be successfully transferred into A. baylyi ADP1, which also has some clinically meaningful. As suggested by the reviewer, phylogenetic tree for amino acid sequences of all Tet(X)s was Moreover, the English grammar in the paper is rather bad and should be improved. I will suggest the authors consult a native speaker to help you remove the flaws from the manuscript.

3.As your described
As suggested by the reviewer, the use of English language throughout the article has been revised.
The "Acinetobacter Bailey ADP1" that appears throughout the article has also been changed to "Acinetobacter Baylyi ADP1".

Other comments:
Line 35: clinical should be clinic.
As suggested by the reviewer, the content of "Introduction" has been modified, the "clinical" has been changed to "clinic", and the modifications can be seen on lines 36.

Line 47: The reference for tet(M) variant was not cited here.
As suggested by the reviewer, the content of "Introduction" has been modified, and the modifications can be seen on lines 47-48 and Ref. 26.

Line 48:enzyme should be enzemy
As suggested by the reviewer, the "enzyme" that appears throughout the article has been changed to "enzymes", and the modifications can be seen on lines 49. The authors should describe this in the text in detail.
As suggested by the reviewer, we sequenced the transconjugants JAT2044 and JAT2091 and found that 5,117-bp circular intermediate (res-ISCR2-ISVsa3-xerD-tet(X3)) forming a minicircle plasmid in the transconjugants JAT2044 to mediates the resistance of the tigecycline. And part of the region of pBDT2091-4 plasmid was re-formed a 27,995-bp plasmid and named pJAT2091. The tigecycline resistance region could be transferred into the recipient strain ADP1 by the mobilization of the conjugative plasmid pBDT2091-6. The modifications can be seen on lines 98-99, 117, 124-136, 173-181 and 226-228.
Line 79: The authors should check 13 consecutive tet(X3) copies carefully to confirm whether it is correct using the original sequencing data.
As suggested by the reviewer, we performed confirmation, but due to the large repeat sequences, we could only confirm that up to 7 consecutive tet(X3) copies results were correct using the raw sequencing data, and finally confirmed that 13 consecutive tet(X3) copies structures were correct by sequence splicing the results. As suggested by the reviewer, the content of "the blaOXA-276 related part including Fig 5" have been deleted.
Line 127-135: It is confusing and should reconstruct the sentence.
As suggested by the reviewer, this sentence has been revised, and the modifications can be seen on lines 144-148.
As suggested by the reviewer, the "brone" that appears throughout the article has been changed to "borne", and the modifications can be seen on lines 163.
Line 157-159: It is confusing and should reconstruct the sentence.
As suggested by the reviewer, this sentence has been revised, and the modifications can be seen on lines 176-178.

Line 173: E.coil should be E.coli
As suggested by the reviewer, the "E.coil" that appears throughout the article has been changed to "E.coli", and the modifications can be seen on lines 192. Line 174: The word "brone" looks like a spelling mistake ?
As suggested by the reviewer, the "brone" that appears throughout the article has been changed to "borne", and the modifications can be seen on lines 163. Lines 204-205: "Nanopore and Illumina reads were combined to produce data for genome assembly were performed with Unicycler version 0.4.3" The complete sequence of the strain in the manuscript is the result of assembly by the unicycler software, however its sequence contains multiple tandem repeat structures, due to the specificity of this structure (the assembly is prone to mismatches), whether the authors used other means (such as checking the long reads raw data or PCR products) to verify the accuracy of these structures?
As suggested by the reviewer, the results of genome assembly with Unicycler version 0.4.3 were quality-checked. We also verify the accuracy of these structures by examining the long read raw data, but due to the high similarity between the tet(X3) variant sequences, it was inconvenient to verify them by PCR amplification.